These studies have further defined the role in inflammatory immune responses of endogenous antioxidant mechanisms and their enhancement by the addition of agents such as 2-mercaptoethanol (2-ME) to stimulated cell cultures. After developing a new assay system which permits the measure of extracellular free thiol concentrations in cell cultures, in situ, studies were performed to define the role of thiols as protective antioxidants. We found that cells exposed to various concentrations of mixed disulfides divide and function in proportion to their capacity to cleave those disulfides to free extracellular thiols. This activity required either a mitogenic or antigenic stimulus. Disulfides could be cleaved by T cells, B cells, macrophages or epithelial cells. The antioxidative function of these extracellular thiols was confirmed by observations that high oxygen tension or oxidizing reagents decreased cell proliferation only after and in proportion to decreasing thiol concentrations. Correlative studies indicate that Gamma-glutamyl transpeptidase is the membrane-bound enzyme responsible for the cleavage of disulfieds to thiols.